Transcriptome analysis highlights the role of ferroptosis in palmitic acid–induced endothelial dysfunction

Abstract Background Palmitic acid (PA) has a lipotoxic effect on blood vessels, leading to endothelial dysfunction and cell death. The underlying mechanisms are not yet fully understood. Aim We sought to investigate the effects of PA on endothelial cells, with an emphasis on ferroptosis. Methods Rat corpus cavernosum endothelial cells (RCCECs) and human umbilical vein endothelial cells (HUVECs) were treated with PA to induce a pattern of cell death, as evidenced by the evaluation of cell viability. The differentially expressed genes were measured via RNA sequencing to reveal potential mechanisms. The intracellular levels of glutathione (GSH), malondialdehyde (MDA), ferrous ion (Fe2+), and reactive oxygen species (ROS) were evaluated using commercial kits. Western blot was performed to determine the expressions of relative proteins. Outcomes At the end of the study period, the evaluated outcomes were cell viability, transcriptome profiles, the expressions of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), as well as levels of GSH, MDA, Fe2+, and ROS. Results PA-induced cell death of RCCECs and HUVECs was demonstrated in a dose- and time-dependent manner. Based on the findings of RNA-sequencing (RNA-seq), enrichment of many biological processes associated with cell cycle and response to stimulus occurred. More importantly, ferroptosis was highlighted in the bioinformatic analysis of both endothelial cells. The levels of intracellular Fe2+, MDA, and ROS were significantly increased following PA exposure while GSH was decreased, suggesting excessive iron accumulation, development of lipid peroxidation, and imbalanced redox homeostasis. Mechanistically, PA decreased the protein expression levels of GPX4 and SLC7A11 in endothelial cells, both of which played crucial roles in ferroptotic cell death. Clinical Translation This study suggests that ferroptosis may be a useful target for novel therapeutic interventions for endothelial dysfunction and cell death in vascular diseases such as erectile dysfunction. Strengths and Limitations In this study, we found that ferroptosis could participate in PA-induced endothelial dysfunction and cell death. A limitation of the study is that it did not shed light on the overall mechanisms of this process. Therefore, further research on the intricate networks of regulating ferroptosis is needed. Conclusion Overall, the occurrence of ferroptosis was demonstrated in the PA-treated HUVECs and RCCECs in this study.


Introduction
Vascular endothelial cells (ECs), also called the vascular endothelium, constitute the interior surface of arteries, veins, and capillaries, which are in direct contact with diverse components and cells of blood. 1 ECs perform many critical functions in controlling tissue homeostasis, including angiogenesis, regulation of vascular tone, blood coagulation, and trafficking of immune cells. 2 More importantly, the impairment of ECs has played an essential role in the pathogenesis of several cardiovascular and endocrinological diseases. For instance, endothelial dysfunction could be an early feature of stroke, diabetes, and atherosclerosis. 3,4 Increased plasma free fatty acids (FFAs) are associated with obesity and type 2 diabetes mellitus and may reduce nitric oxide production, triggering the onset of endothelial dysfunction. 5,6 Palmitic acid (PA) is one of the most abundant FFAs in the human body and often occurs in the daily diet. PA has been reported to lead to aggravated apoptosis of vascular ECs, which has attracted increasing attention owing to its lipotoxic effect on blood vessels. 6 Recently, rapid progress has occurred in understanding of related mechanisms, such as excessive generation of reactive oxygen species (ROS), impaired insulin signaling, and upregulation of inflammatory signaling. Therefore, modulation of the involved pathways may protect against endothelial dysfunction and provide a favorable cardiovascular outcome.
Ferroptosis, a novel modality of programmed cell death dependent on iron, is characterized by an overload of lipid peroxidation products and excessive iron accumulation, which is generally accompanied by increased lipid peroxide and ROS production. 7 It has been revealed that system X c − and glutathione peroxidase 4 (GPX4) are critically important in the suppression of ferroptosis. 8 System X c − is a transmembrane protein complex containing the subunit solute carrier family 7 member 11 (SLC7A11), whose blockage can lead to the depletion of glutathione (GSH). In addition, GPX4 is the major enzyme catalyzing the reduction of GSH, protecting cells against ferroptosis by converting lipid peroxides into nontoxic lipid alcohols. 9 Recent studies have demonstrated that ferroptosis has been linked to various pathologies associated with tissue damage and subsequent cell loss, such as tumors, ischemia-reperfusion injury, and neurological diseases. 8 However, the role of ferroptosis in the dysfunction and cell death of ECs stimulated by PA remains to be elucidated.
In this study, we aimed to investigate the potential mechanisms of PA-induced endothelial dysfunction and cell death, determining whether ferroptosis is involved in this process. Our findings have shown that PA can lead to ferroptotic cell death, providing new insight into the role of ferroptosis in related diseases.

RNA isolation, library preparation, and RNA sequencing
Total RNA from PA-treated or untreated ECs was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The libraries were constructed with the mRNA-seq Lib Prep Kit (Cat# RK20302, ABclonal, Wuhan, Hubei, China) and sequenced on an Illumina NovaSeq 6000 platform using 150-bp pairedend sequencing mode.
The raw files were assessed for initial quality using FastQC (version 0.11.7), trimmed for adapter sequences using Cutadapt (version 2.7), and aligned to the human (GRCh38.102/hg38) and rat (Rnor_6.0.102/rn6) reference genome using Hisat2 (version 2.0.5). Transcript assembly was carried out using Stringtie. The gene abundance was expressed as the fragments per kilobase of transcript per million reads mapped. The differentially expressed genes (DEGs) were analyzed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to identify enriched pathways and potential functions.

Cell viability analysis
HUVECs and RCCECs were seeded in a 96-well plate format for 24 hours and treated with PA (up to 1.0 mM) at 37 • C and 5% CO 2 . The cell viability was measured using a Cell Counting Kit-8 (CCK-8; Cat# KGA317, Keygen Biotech, Nanjing, China), with CCK-8 solution (10 μL/well) added to the cells. After a 2-hour incubation at 37 • C and 5% CO 2 , absorbance at 450 nm was determined with a microplate reader (ThermoFisher Scientific, Waltham, MA, USA).

Determination of ROS production
Intracellular ROS production was determined using an ROS assay kit (Cat# S0033; Beyotime Biotech, Shanghai, China). The ECs were exposed to the indicated conditions and then incubated with DCFH-DA (10 μM) for 30 min at 37 • C and 5% CO 2 . After being washed with phosphate-buffered saline (PBS), the cells were observed under a fluorescence microscope and the intensity was quantified using ImageJ software.

Measurement of Fe 2+ , MDA, and GSH
According to the manufacturer's protocols, the intracellular values of ferrous ion (Fe 2+ ), malondialdehyde (MDA), and GSH in ECs were measured using FerroOrange (Cat# F374;

Statistical analysis
The data are presented as mean (SD). Comparisons between 2 groups were made with the unpaired Student t-test. Oneway ANOVA followed by Tukey's multiple comparisons or Kruskal-Wallis test were used to determine an overall difference among various groups. Statistical analyses were performed using GraphPad Prism, version 9.0 (GraphPad Software, San Diego, CA, USA). P values less than 0.05 were considered significant.

PA induced EC death
To explore the effect of PA on the cell death of ECs, different concentrations of PA were used to treat HUVECs and RCCECs at different time points. The CCK-8 assay showed that compared with negative controls cell viability was decreased after PA exposure, depending on its concentrations and duration ( Figure 1). Specifically, PA (0.25 mM, 24 hours) caused approximately 60% and 50% of cell death in HUVECs and RCCECs, respectively.

Ferroptosis was enriched based on the RNA-seq analysis
To identify the molecular mechanism involved in PA-induced cell death, RNA-seq analysis was performed to determine the DEGs in 2 types of ECs between the PA-treated group and negative controls (Figure 2). A total of 1679 DEGs were identified in HUVECs and 979 in RCCECs, suggesting that HUVECs and RCCECs are sensitive to PA exposure. GO functional analysis indicated that DEGs were enriched in biological processes associated with the cell cycle and response to stimulus, including response to organic substances, regulation of cell cycle, and chromosome segregation ( Figure 3). Furthermore, molecular binding was also suggested to participate in the cell damage caused by PA.
The enrichment analysis of KEGG demonstrated that the DEGs significantly mapped to multiple cellular pathways of inflammation, apoptosis, and metabolism ( Figure 4). Notably, ferroptosis was predicted in both EC cell types, indicating its essential function in PA-induced cell death. Meanwhile, ferroptosis-related pathways such as GSH metabolism and p53 signaling pathways were also enriched in the KEGG analysis.

PA triggered the ferroptosis of ECs
To determine whether PA can induce the ferroptosis of ECs, we sought to detect the levels of lipid peroxidation and intracellular ROS after treatment with 0.25 mM PA. We observed that ROS production and levels of MDA were significantly increased following PA exposure whereas GSH was decreased in both ECs, suggesting the development of lipid peroxidation and imbalanced redox homeostasis ( Figure 5A-D). Moreover, PA induced a notable increase of intracellular Fe 2+ levels compared to negative controls ( Figure 5E). These findings were consistent with established knowledge regarding ferroptosis under specific biological contexts.
Because the Cyst(e)ine/GSH/GPX4 axis represents a canonical ferroptosis-controlling pathway, we investigated the expression of related proteins. As expected, the protein expression level of GPX4 was significantly reduced in HUVECs and RCCECs treated with PA (0.25 mM, 24 hours compared with untreated cells (Figure 6). In addition, the protein expression of SLC7A11, a key component of the system X c − cystine/glutamate antiporter responsible for cellular uptake of extracellular cystine in exchange for intracellular glutamate, was also decreased by PA treatment, even at a lower concentration. Collectively, these data suggested that PA triggered ferroptosis in HUVECs and RCCECs.

Discussion
Endothelial cell dysfunction, injury, and death remain the leading causes of diverse pathological settings such as cardiovascular diseases, erectile dysfunction, and ischemiareperfusion injuries. [12][13][14] A growing number of published studies in this field suggest that the accumulation of lipids can pose a threat to the normal function of ECs. 15,16 Proinflammatory molecules can be generated in ECs by saturated FFAs such as myristic acid (C14:0), PA (C16:0), and stearic acid (C18:0), which may be mediated through the alteration of cell membrane properties to activate Tolllike receptors. 17 FFAs are also principal sources of ROS accumulation, which leads to oxidative stress. Moreover, saturated FFAs can promote apoptosis of ECs via nuclear factor-kappaB activation. 18 Recent studies have focused on the role of ferroptosis, a newly discovered form of programmed cell death, in certain pathological contexts. Mounting evidence has demonstrated that ferroptosis has unique network mechanisms and functions, which are different from necrosis, apoptosis, and autophagy. Dysregulated lipid metabolism, especially unrestrained phospholipid peroxidation, has been the hallmark of ferroptosis. 8 In the present study, we demonstrated the occurrence of ferroptosis within HUVECs and RCCECs exposed to PA, providing preliminary evidence for the role of ferroptosis in endothelial dysfunction and cell death induced by high levels of FFAs ( Figure 7). Saturated FFAs are inevitable in Western dietary patterns, particularly in animal-based foods. For example, PA and stearic acids are predominant in butter, palm kernel oil, meat,  and dairy products. Compared with unsaturated FFAs, they are thought to be associated with a significantly increased risk for cardiovascular diseases. 19 Reducing the consumption of saturated fat has been prioritized in current public health dietary recommendations for the improvement of cardiovascular health. 20 It has been established that elevated levels of plasma FFAs often cause a detrimental imbalance in cell metabolism. 5 Previous studies have shown that PAinduced EC death has been involved with various signaling pathways, including autophagy, apoptosis, and necroptosis. 18,21 However, the underlying mechanisms are not yet fully understood. In this study, HUVECs and RCCECs were used to identify other possible forms of cell death involved in this process. The cell viability of PA-induced ECs was significantly reduced compared with that of untreated cells, indicating that EC death was successfully elicited. The findings of RNAseq suggest the involvement of ferroptosis in HUVECs and RCCECs following the stimulation of PA. Wang et al. reported the development of ferroptosis in cardiomyocytes exposed to PA, in which HSF1 may serve as a key defender against ferroptotic cell death. 22 Moreover, PA-induced ferroptosis was also observed in numerous types of cells, including vascular smooth muscle cells and hepatocytes. 23,24 Therefore, PA-mediated ferroptosis in endothelial dysfunction and cell death was taken into consideration in the present study.
Extensive investigations of ferroptosis over the past 20 years have rapidly broadened our understanding of this form of cell death, which is mainly executed by phospholipid peroxidation resulting from impaired metabolism of iron, ROS, and phospholipids. Initially, mechanisms governing ferroptosis are centered around cysteine and GSH metabolism as well as the activity and stability of GPX4. 25 In addition, the system X c − is an amino acid antitransporter composed of 2 subunits, SLC7A11 and SLC3A2, which affect the action of glutathione peroxidases and the synthesis of GSH. 26 The downregulation of system X c − and GPX4 has been implicated in the ferroptotic response of cancer cells. 25,27 In this study, expression of GPX4 and SLC7A11 was reduced in the ECs treated with PA, suggesting the promotion of ferroptosis in this setting. After the stimulation of PA, HUVECs and RCCECs produced excessive ROS and MDA and significantly inhibited the biosynthesis of GSH, which reflected the sustained activation of lipid peroxidation and oxidative stress. It has been reported that zinc oxide nanoparticles can induce ferroptosis of HUVECs, as evidenced by the assessment of ROS production, lipid peroxidation, and cell viability. 28 Additionally, ferroptosis depends on macroautophagy/autophagy and is mediated by ferritinophagy in such a context. As the biological functions of ferroptosis have been depicted to some extent, its pharmacological modulation, via either inhibition or induction, may yield promising clinical outcomes for certain diseases. For example, it has been demonstrated that metformin and GSK-J4 may ameliorate lipotoxicity to cardiomyocytes via ferroptosis. 23,29 Quercetin, which decreases the expression of ATF3, could alleviate acute kidney injury via inhibition of ferroptosis. 30 Overall, ferroptosis might play an important role in PA-treated endothelial dysfunction and cell death.
Several principal limitations should be acknowledged in our study. First, endothelial dysfunction and cell death owing to acute PA infusion differ from the chronic process in the human body, although elevated levels of plasma FFAs are a remarkable risk factor for vascular diseases. Second, the lack of other types of ECs and animal model studies may not illustrate the entire mechanisms of ferroptosis under these pathological conditions. Third, ferroptosis can be regulated via intricate networks that are more diverse than originally supposed. Recent advances have uncovered GPX4independent mechanisms of ferroptosis surveillance, including NADPH/FSP1/CoQ10 and GCH1/BH4 pathways. 8 More evidence is required to address these issues for potential discoveries in this exciting field.

Conclusions
In summary, ferroptosis occurrence was implicated in the PA-treated HUVECs and RCCECs based on the findings of RNA-seq and related biochemical investigations. Further research should be directed at ferroptosis in the development of endothelial cytotoxicity and dysfunction, which might be a potential target for novel therapeutic interventions.

Funding
The work was supported by the National Natural Science Foundation of China (grant no. 81971379).

Conflicts of interest:
The authors declare no potential conflicts of interest.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files, which are available on reasonable request. RNA-Seq data have been made available in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/ geo/) under the accession number GSE205913.